Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis.

Rapid and reproducible method is described here for the purification of the 43 kDa fimbrial protein from P. gingivalis by preferential fractionation in the presence of 1% SDS and 0.2M of a bivalent cation at pH 6.5. Homogeneity of the purified 43 kDa was confirmed by SDS-PAGE and Western blot analysis using monoclonal and polyclonal antibodies raised against this protein. Amino acid composition and the amino acid sequence of the first 30 amino acid residues of the purified fimbriae are consistent with the composition and sequence predicted from the cloned gene of the fimbrial subunit. Circular dichroism spectra shows high levels of beta-sheet structure. The purified 43 kDa polymer shows fimbriae-like morphology under the electron microscope. Ultrastructural localization of the 43 kDa protein by the immunogold technique revealed specific labeling of the fimbriae with a diameter of approximately 3.5 to 5.0 nm. Localization of this protein suggest that the 43 kDa component is a fimbrial subunit.     

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of ¹³C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.     

Nanoemulsion based gel for transdermal delivery of meloxicam: Physico-chemical,mechanistic investigation

AimsThe aim of the present investigation was to develop a nanoemulsion (NE) gel formulation for the transdermal delivery of meloxicam (MLX) in order to ensure maximum controlled and sustained drug release capacity.Main methodsThe MLX containing NE gel was prepared and characterized for particle size, zeta potential, pH, rheology, in vitro drug release, in vitro skin permeation, and in vitro hemolysis. Differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) of MLX-NE gel treated rat skin was performed to investigate the skin permeation mechanism of meloxicam from NE gel. Skin permeation potential of the developed gel formulation was assessed using confocal laser scanning microscopy (CLSM). The in vivo toxicity of MLX-NE gel was assessed by histopathological examination in rat. The rat paw edema test was performed to evaluate the anti-inflammatory activity of MLX-NE gel.Key findingsPercutaneous absorption studies demonstrated a higher permeation of meloxicam from NE gel, than the drug solution. FTIR and DSC studies supported stratum corneum lipid extraction as a possible penetration enhancer mechanism for MLX-NE gel. CLSM studies confirmed the permeation of the NE gel formulation to the deeper layers of the skin (up to 130 μm). MLX-NE gel turned out to be non-irritant, biocompatible, and provided maximum inhibition of paw edema in rats over 24 h in contrast to MLX solution.SignificanceThe nanoemulsion gel formulation may hold promise as an effective alternative for the transdermal delivery of meloxicam.     

Taxonomic study of spongy spumellarian Radiolaria with three and four coplanar spines or arms from the middle Carnian (Late Triassic) of the Köseyahya nappe (Elbistan, SE Turkey) and other Triassic localities

The present article is a taxonomic study of all spongy spumellarian radiolarian taxa with three and four coplanar spines or spongy arms occurring in the middle Carnian from the Köseyahya section, near the town of Elbistan, SE Turkey. This fauna is characteristic of the Tetraporobrachia haeckeli radiolarian Zone, and comes from an 8 m thick succession of clayey-cherty limestones occurring at the lower part of the section. In addition, a few species from the Middle and Upper Triassic from other areas have been also included in this study to improve some generic diagnoses, and to better comprehend the diversity and evolutionary trends of some genera, subfamilies and families. The taxonomy at the generic and suprageneric levels is based primarily on the types of microsphere. This new approach allowed new taxonomic arrangements of genera and suprageneric units, and suggested new phylogenetic relationships among these radiolarians and between them and younger radiolarians. The authors discuss and describe 69 species, of which 37 are new, and 14 genera, of which three are new (Paraparonaella, Pseudangulobracchia, and Ropanaella). The genus Triassoastrum and others are reinterpreted. All genera studied are assigned to five subfamilies, of which two are new (Tetrapaurinellinae and Triassocrucellinae), and two families (Tritrabidae and Veghicycliidae). Nine species in open nomenclature are also illustrated.     

The Relative Expression of Mig6 and EGFR Is Associated with Resistance to EGFR Kinase Inhibitors

The sensitivity of only a few tumors to anti-epidermal growth factor receptor EGFR tyrosine kinase inhibitors (TKIs) can be explained by the presence of EGFR tyrosine kinase (TK) domain mutations. In addition, such mutations were rarely found in tumor types other than lung, such as pancreatic and head and neck cancer. In this study we sought to elucidate mechanisms of resistance to EGFR-targeted therapies in tumors that do not harbor TK sensitizing mutations in order to identify markers capable of guiding the decision to incorporate these drugs into chemotherapeutic regimens. Here we show that EGFR activity was markedly decreased during the evolution of resistance to the EGFR tyrosine kinase inhibitor (TKI) erlotinib, with a concomitant increase of mitogen-inducible gene 6 (Mig6), a negative regulator of EGFR through the upregulation of the PI3K-AKT pathway. EGFR activity, which was more accurately predicted by the ratio of Mig6/EGFR, highly correlated with erlotinib sensitivity in panels of cancer cell lines of different tissue origins. Blinded testing and analysis in a prospectively followed cohort of lung cancer patients treated with gefitinib alone demonstrated higher response rates and a marked increased in progression free survival for patients with a low Mig6/EGFR ratio (approximately 100 days, P = 0.01).     

Purification and Characterization of Rat Parotid Glycosylated,Basic and Acidic Proline-Rich Proteins

Abstract

A unique family of proline-rich proteins (PRPs) is induced in rats following prolonged isoproterenol treatment. PRPs can be divided into glycosylated (GPRP), basic (BPRP) and acidic (APRP) proline-rich proteins based on their physicochemical characteristics. Inducible rat parotid PRPs were isolated from aqueous extracts of parotid glands of isoproterenol-treated animals by sequential chromatography on columns of DEAE-Sepharose CL-6B, Sephadex G-100 and FPLC on Suprose-12 column. The GPRP showed a single homogeneous band on sodium dodecylpolyacrylamide gel electrophoresis with an estimated molecular weight of approximately 220,000. Compositional analysis of GPRP revealed that this protein contained 19.7% glutamic acid/glutamine, 28.2% proline and 9.5% glycine, and 44% carbohydrate, consisting of fucose (2.81g/100g), mannose (9.78g/100g), galactose (9.29g/100g), N-acetylglucosamine (18.03g/100g) and N-acetylgalactosamine (3.90g/100g). Basic PRPs consisted of a family of proteins with estimated molecular masses ranging from 14–45 kDa. These proteins contained 42.6% proline, 20.65% glutamic acid/glutamine and 21.33% glycine. Acidic PRPs also comprised of a family of metachromatically stained ladder of 40–60 kDa containing 29.1% proline, 21.5% glutamic acid/glutamine and 17.8% glycine. APRP were heavily glycosylated containing N-acetylglucosamine (6.34g/100g), N-acetylgalactosamine (19.04g/100g) and glucuronic acid (38.08g/100g).     

IL‐18 Cytokine Levels Modulate Innate Immune Responses and Cryptosporidiosis in Mice

IL‐18 is known to play a key role limiting Cryptosporidium parvum infection. In this study, we show that IL‐18 depletion in SCID mice significantly exacerbates C. parvum infection, whereas, treatment with recombinant IL‐18 (rIL‐18), significantly decreases the parasite load, as compared to controls. Increases in serum IFN‐γ levels as well as the up‐regulation of the antimicrobial peptides, cathelicidin antimicrobial peptide and beta defensin 3 (Defb3) were observed in the intestinal mucosa of mice treated with rIL‐18. In addition, C. parvum infection significantly increased mRNA expression levels (> 50 fold) of the alpha defensins, Defa3 and 5, respectively. Interestingly, we also found a decrease in mRNA expression of IL‐33 (a recently identified cytokine in the same family as IL‐18) in the small intestinal tissue from mice treated with rIL‐18. In comparison, the respective genes were induced by IL‐18 depletion. Our findings suggest that IL‐18 can mediate its protective effects via different routes such as IFN‐γ induction or by directly stimulating intestinal epithelial cells to increase antimicrobial activity.     

Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design,synthesis, biological investigation and docking studies

In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC₅₀ value of 1.06 µM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.     

A study of the impact of oral health on the quality of life of older people in the UK- findings from a National Survey.

Objectives The aim of this study was to was to determine whether older adults perceive oral health as affecting their life quality and to identify variations in impacts in relation to socio-demographic factors, dental service utilisation and method of payment. Design This study formed part of the Office for National Statistics Omnibus Survey, which utilised a random probability sample of addresses from the British Postcode Address File (PAF). Setting 3,000 homes were selected from one hundred post sectors across Britain. Respondents were interviewed in their homes about how oral health affects their quality of life. Subjects 454 older people (aged 65 and over) took part in the survey. Main outcome measures The study group perceived oral health as impacting on their quality of life in general (negative and/or positive impact) (70%, 318), enhancing (53%, 241) and detracting (44%, 199) from their life quality. The most frequently perceived way in which oral health affects life quality was its effect on eating and comfort. Older people from higher socio-economic groups reported that oral health had a greater impact on their quality of life in general (positive and/or negative impacts), (OR=1.77,95% CI= 1.22,2.78) and specifically, enhancing their quality of life (OR=1.52, 95% CI=1.01,2.30). Those who reported attending the dentist within the last year perceived that their oral health enhanced their life quality (OR=1.55, 95% CI=1.01,2.38). Conclusions Socio-economic background and dental attendance pattern are associated with how older people perceived oral health as affecting quality of life. These findings may have implications for promoting regular dental attendance and auditing dental services for older people.     

Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance

Background

The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies.

Methodology/Principal Findings

In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo.

Conclusions/Significance

The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.